Malignant cells in tumours of B-cell origin account for 0.1% to 98% of the total cell content, depending on disease entity. Recently, gene expression profiles (GEPs) of B-cell lymphomas based on microarray technologies have contributed significantly to improved sub-classification and diagnostics. However, the varying degrees of malignant B-cell frequencies in analysed samples influence the interpretation of the GEPs. Based on emerging next-generation sequencing technologies (NGS) like tag sequencing (tag-seq) for GEP, it is expected that the detection of mRNA transcripts from malignant B-cells can be supplemented. This study provides a quantitative assessment and comparison of the ability of microarrays and tag-seq to detect mRNA transcripts from malignant B-cells. A model system was established by eight serial dilutions of the malignant B-cell lymphoma cell line, OCI-Ly8, into the embryonic kidney cell line, HEK293, prior to parallel analysis by exon microarrays and tag-seq.CD74, HLA-DRA, and BCL6 - was analysed. It was noticed that at least 5%, 13% and 22% sample purity respectively was required for detection of the three genes by exon microarray whereas at least 2%, 4% and 51% percent sample purity of malignant B-cells were required for tag-seq detection.We identified 123 and 117 differentially expressed genes between pure OCI-Ly8 and HEK293 cells by exon microarray and tag-seq, respectively. There were thirty genes in common, and of those, most were B-cell specific. Hierarchical clustering from all dilutions based on the differentially expressed genes showed that neither technology could distinguish between samples with less than 1% malignant B-cells from non-B-cells. A novel statistical concept was developed to assess the ability to detect single genes for both technologies, and used to demonstrate an inverse proportional relationship with the sample purity. Of the 30 common genes, the detection capability of a representative set of three B-cell specific genes - A sample purity-dependent loss of the ability to detect genes for both technologies was demonstrated. Taq-seq, in comparison to exon microarray, required slightly less malignant B-cells in the samples analysed in order to detect the two most abundantly expressed of the selected genes. The results show that malignant cell frequency is an important variable, with fundamental impact when interpreting GEPs from both technologies. Malignant cells in tumour tissues of B-cell origin account for 0.1% to 98% of the total cell content depending on the specific disease entity . VaryingNlaIII and MmeI, to generate cDNA tags that are reverse transcribed from mRNA transcripts, by cutting from the 3′-end CATG to the poly(A)-tail and 17–19 bp downstream of the first restriction enzyme site [The Affymetrix GeneChip Human Exon 1.0 ST Array is a dense microarray designed for GEP, featuring 6.5 million probes corresponding to known targets and predicted exons spanning the entire human genome . The proyme site ,11. ClonThe present study is based on a model system, where malignant B-cells were serially diluted into embryonic kidney cells at eight different cellular frequencies illustrating varying levels of malignant B-cell sample purities. Total RNA from each of the cell populations was tested in parallel to generate data, both by exon microarrays using the Affymetrix GeneChip Human Exon 1.0 ST platform and tag-seq using the Illumina Genome Analyzer platform, as well as RT-qPCR validation of three selected genes. The goal for this work was to develop a quantification method that could be used for assessing and comparing the ability to detect malignant B-cell transcripts based on exon microarray and tag-seq as a function of sample purity.To compare the detection abilities, serial dilutions of eight samples of mixed cell populations from the human malignant B-cell line, OCI-Ly8, into the human embryonic kidney cell line, HEK293, at the cellular frequencies 0%, 0.5%, 1%, 5%, 10%, 20%, 30%, and 100% malignant B-cells, were prepared by fluorescent activated cell sorting (FACS). This dilution scheme was chosen to demonstrate resolution detection for small amounts of malignant B-cells. Total RNA from all samples was prepared and analysed using the two technologies in question. Overall, mRNA expression levels of 17,816 RefSeq transcripts and full-length mRNA transcripts using the core probes of the exon microarray were determined, and the number of unique genes detected above background (DABG) was 11,112. In parallel, exactly the same total RNA from all samples was used to prepare small tags of 17 bp cDNA sequences, which yielded 0.5-2.8 million reads per sample  [CD20 (MS4A1), CD45 (PTPRC), CD52, and CD53, CD74, CD79A and B), human leukocyte antigen molecules (HLADRA), transcriptional regulators , B-cell-specific scaffold proteins (BANK1), creatine kinase isoenzymes (CKB), deubiquitinating enzymes (UCHL1), actin-binding proteins (LCP1), glycosyltransferases (ST6GAL1), uncharacterised proteins (C13orf18), and signal transduction molecules (GCET2). The full lists of differentially expressed genes are given in Additional files Differentially expressed genes were determined between the pure OCI-Ly8 and HEK293 cell line samples using a false discovery rate (FDR) of less than 5%. These settings identified 123 genes by exon microarray and 117 genes by tag-seq. However, only 30 genes were concordant, as indicated in Figure For both exon microarray and tag-seq, hierarchical clustering of all samples based on the 123 and 117 differentially expressed genes resulted in three individual concordant clusters  and therefore only borderline expressed. This indicates that very few false-positive B-cell specific mRNA transcripts were detectable in the pure HEK293 cell line sample. Of the 117 differentially expressed genes between 100% and 0% samples detected by tag-seq, 61 genes were expressed more highly in the 100% B-cell sample. All had counts equal to zero except for 4 genes  that had counts of 2, 1, 3 and 1 respectively in the pure HEK293 cell lines, demonstrating very few, if any, false-positive B-cell specific mRNA transcripts using the tag-seq technology.Of the 123 genes differentially expressed between the 100% and 0% samples detected by exon microarray, we identified 79 B-cell specific genes that were expressed more highly in the 100% B-cell sample. To see whether these 79 B-cell specific mRNA transcripts were present in the background of the non-B-cells, the proportion of B-cell specific mRNA transcripts with DABG was plotted as a function of sample purity  of exon microarray and tag-seq were determined. By regression of the mRNA expression levels of the B-cell specific differentially expressed genes versus sample purity under the assumption of a common background as intercept and gene-individual slopes, the IDL was determined for both technologies Figure . For exoThe ability to detect genes was quantified by plotting the MDL as a function of sample purity Figure . For botCD74, HLA-DRA, and BCL6 are all transcripts of importance in B-cell development, and they were among the 30 concordant differentially expressed genes between exon microarray and tag-seq, they were selected for comparison and verification of the MDL as a function of sample purity  increases with sample purity Figure . Both exquadratic mean variance negative binomial model, i.e. NB2, when detecting differentially expressed genes, whereas we used the linear meanvariance negative binomial model, i.e. NB1, when analysing the IDL. NB1 is not supported in the edgeR package but we discovered that NB1 was sufficient for the negative binomial regression by residual plots  in a humidified atmosphere supplemented with 5% CO2. Cells were harvested in the exponential growth phase and sorted by a BD FACSAria II Cell Sorter  to obtain eight cell populations, diluting OCI-Ly8 into HEK2932 in the cellular concentrations 0%, 0.5%, 1%, 5%, 10%, 20%, 30%, and 100% of OCI-Ly8 and subsequently stored in Trizol Reagent . Cell line identity was analysed using 0.2 ng/μl extracted RNA from the cell lines as template in a multiplex PCR using the AmpFISTR Identified PCR amplification kit . A fragment analysis of the amplified PCR product was performed by capillary electrophoresis by Eurofins Medigenomix GmbH, Applied Genetics, Germany. The resulting FSA files were analysed using the Osiris software (http://www.ncbi.nlm.nih.gov/projects/SNP/osiris/) given the following STR profiles: OCI-Ly-8: D5S818-4 , D13S317-2 , D7S820-1 , D16S539-2 , VWA-3 , TH01-2 , AMEL-4 , TPOX-3 , CSF1PO-1 , and HEK293: D5S818-4 , D13S317-2 , D7S820-1 , D16S539-2 , VWA-3 , TH01-2 , AMEL-4 , TPOX-3 , CSF1PO-1 .The cell line, OCI-Ly8, provided by Dr. Hans Messer and Dr. Andreas Rosenwald was established at Ontario Cancer Institute (OCI). OCI-Ly8 was established from a patient with diffuse large B-cell lymphoma . This cemirVana miRNA Isolation Kit  according to the manufacturer’s protocol. The quantity of extracted total RNA was measured on a NanoDrop ND-1000 spectrophotometer , and the quality of the total RNA was assessed using the Agilent RNA 6000 Nano kit and Nano Series II assay in combination with the Agilent 2100 Bioanalyzer . Total RNA was stored at −80°C until use.Total RNA was extracted from the eight cell populations using the Trizol Reagent and purified using the Exon microarray data were generated using 100 ng of total RNA. Ambion WT Expression Kit  generated amplified and biotylated sense-strand cDNA from the total RNA according to the manufacturer’s protocol. Afterwards, single-strand cDNA was fragmented by UDG (uracil glycosylase) and APE1 (apurinic/apyrimidinic endonuclease) and biotin-labelled using the GeneChip Terminal Labeling and hybridisation Kit  according to the manufacturer’s protocol. 5 μg of biotin-labelled cDNA were incubated with the Affymetrix GeneChip Human Exon 1.0 ST array at 45°C for 17 hours for hybridisation. Following hybridisation, non-specific bound material was removed by washing with the GeneChip Hybridisation, Wash and Stain kit  and the GeneChip Fluidics Station 450 . Detection of bound material was performed by scanning the array using the GeneChip Scanner 7G .NlaIII  to generate cDNA fragments from the most 3′-end CATG to the poly(A) tail. cDNA without the NlaIII recognition site was removed by washing. A biotinylated adaptor containing the recognition site for the restriction enzyme MmeI was linked to the 5′-end of the digested double stranded cDNA. Next, tags of 17 bp of double stranded cDNA were released from the Oligo-(dT)25 beads by MmeI . Tags were purified from a polyacrylamide gel by phenol-chloroform extraction and bound to Streptavindin beads. A second adapter containing an identification key of 3 bp unique for each sample was ligated to the tags. The adaptor-ligated tags from all samples were pooled and amplified by PCR. The PCR products were purified by excision from a 6% polyacrylamide gel , followed by elution, ethanol precipitation and re-suspension . The amount of adaptor-ligated tags was quantified using the NanoDrop ND-1000 spectrophotometer . The sample was stored at −20°C until sequencing. The sequencing procedure was carried out according to the manufacturer’s protocol. Adaptor-ligated tags from all samples were sequenced in an individual lane of a flow cell by the Solexa/Illumina Whole Genome Sequencer 1G analyser. Clustering and sequencing were performed with reagents from the cluster generation kit and sequencing kit .For tag preparation, 2 μg of total RNA were incubated with magnetic Oligo-(dT) beads to capture the polyadenylated RNA fraction. Bound to the beads, total RNA was reverse transcribed into double-stranded cDNA using the SuperScript II kit . The double-stranded cDNA was digested with the restriction enzyme RT-qPCR data were generated using total RNA reverse transcribed into cDNA by the SuperScript III First-Strand Synthesis Supermix  and TaqMan reagents and assays  according to the manufacturer’s protocol. 500 ng total RNA was mixed with 50 μM Oligo(dT) primer, 50 ng/μl random hexamer primer, and Annealing buffer. The sample was incubated in the thermal cycler at 65°C for 5 minutes to unfold loops together with secondary structures. Following incubation, 2x First-Strand Reaction mix and SuperScript III/RNaseOUT Enzyme mix were added to the sample, and the sample was incubated in the thermal cycler at 25°C for 5 minutes, 50°C for 50 minutes, and then 80°C for 5 minutes. The cDNA was used directly in RT-qPCR or stored at −20°C.For RT-qPCR, 2x TaqMan Universal Master mix without AmpErase UNG  and 20x TaqMan gene expression assay was mixed. The cDNA product was diluted 10 times, and 4 μl of the cDNA product was used as template. The cDNA product was amplified using a two-step thermal profile: 95°C for 10 minutes, followed by 50 cycles at 95°C for 15 seconds and 60°C for 1 minute.CD74 (Hs00959496_m1), HLA-DRA (Hs00219576_m1), and BCL6 (Hs00153368). To correct for sample-to-sample variations in efficiencies and errors in quantification, the TaqMan Pre-Developed endogeneous control assay , TBP (333769F), were used for normalisation. Amplified mRNA was detected by the Mx3000P QPCR instrument .The RT-qPCR was performed using cDNA product and the TaqMan gene expression assays  specific for http://bcgc.lbl.gov/cdfFiles/HuEx-1_0-st-v2,A20071112,EP/HuEx-1_0-st-v2,coreR3,A20071112,EP.cdf.CEL-files were generated by the Affymetrix GeneChip Command Console Software (AGCC) and deposited at the NCBI Gene Expression Omnibus (GEO) under accession number GSE40311. The .CEL-files were imported into Bioconductor using the aroma.affymetrix package. The .CEL-files were background corrected and quantile normalised by the RMABackgroundCorrection and QuantileNormalization functions from the aroma.affymetrix package . Core prThe Illumina Pipeline software version 1.3 was used for image analysis and base calling, omitting chastity filtering, otherwise using default settings. Data were imported into the CLC Genomics Workbench 4.5 and sequence error corrected by the SAGEScreen method. The first 17 bp of the tag sequences were extracted from the output file according to the second adaptor containing the identification key for each sample. The unique tags were sorted and counted for each sample using the CLC Genomics Workbench 4.5 and exported to .txt-files. These text files were imported into Bioconductor. Due to a large library-to-library variation in the amount of obtained tags the set of tags in the libraries were randomly sub-sampled without replacement to equal the size of the smallest library.ftp://ftp.ensemble.org on both the coding and non-coding strands using Bowtie version 0.12.7 [Each unique tag sequence was annotated by mapping the reads with the anchoring NlaIII restriction site to the ENSEMBL Homo Sapiens cDNA reference transcriptome (release 63) downloaded from n 0.12.7 . The setP-values were false discovery rate (FDR) corrected by the Benjamini-Hochberg procedure and 0.05 was used as significance level.All statistical analyses were done with R Version 2.13.2 (64 bit Windows version) and a number of Bioconductor packages  as descrYgi, for the g’th gene out of G genes and the i’th sample out of two samples is N - distributed. Then, under the null hypothesis of no difference in gene expression of gene g between sample 1 and 2, Yg1 − Yg2, is N -distributed. IfSince no replicates were present for the pure cell lines, it was assumed for exon microarrays that the expression value, P-values of the z-scores zg = (Yg1 − Yg2)/s can be used to test the hypothesis of no difference in gene expression levels. The P-values of the z-scores can be FDR corrected by the Benjamini-Hochberg procedure as implemented in the Bioconductor package fdrtool.this means that Ygi, of a gene g in sample i as a quadratic mean-variance negative binomial distributed random variableThe Bioconductor package edgeR is designed for differential expression analysis of count data and is an implementation of the methods developed by Robinson and Smyth . As recoP-values for differentially expressed genes were determined using an exact test.where Mi is the library size, pgi is the relative abundance of gene g, and ϕg is the dispersion parameter. When the negative binominal distribution was fitted, a common dispersion for all tags was estimated using the Cox-Reid profile-adjusted likelihood method. FDR corrected P-values for the hypothesis that each exon belongs to the background. The .PGF and .CLF files were used for the exon array, and for the background correction, the antigenomic background probes were used. Summarising to gene level was performed as follows [P-value was below 0.005. If half or more than half of the exons were present, the gene was assumed to be present in that sample, or otherwise it was absent.In order to determine genes detected above background, the DABG algorithm of Affymetrix Power Tools (APT)  version  follows : For eacA hierarchical cluster analysis was performed on the differentially expressed genes. The hierarchical cluster analysis was based on the R-function hclust using complete linkage as the agglomeration method and Eucledian distance as dissimilarity measure.b, the random variable Y(t) denote a measurement of the analyte at concentration t ≥ 0 and the statistical confidence is chosen to be 95%. Then, the IDL is defined asThe instrumental detection limit (IDL) is defined as the analyte concentration required to produce a signal that is distinguishable from the blank level with a particular statistical confidence . Formallt, should be understood as the light intensity arising from a particular mRNA transcript hybridised to the array. It is normally assumed that this light-intensity is directly proportional to the number of mRNA transcripts in the sample. The coefficient of proportionality depends on e.g. the array type and reaction efficiency. Hence, for exon microarrays, the following measurement model is assumedFor exon microarray, the concentration, ε(t) is assumed to be N -distributed. In this casewhere p, 0 ≤ p ≤ 1, is denoted as the ratio between the tumour and normal samples, the following measurement model is assumedThe method detection limit (MDL) will in practice depend on the sample purity. If p in the following wayThen, it is straightforward to calculate the MDL as a function of the sample purity k’th transcript, tkxi where 0 = x1 ≤ ⋯ ≤ xn = 1, then, for exon microarray, this means that b, tk and σ can be estimated by the linear regression modelThis shows that the MDL is inversely proportional to the sample purity. The IDL can be estimated by dilution curves as follows: Let the gene expression be measured at the following dilutions of the εki ~ N. This is done by means of least squares regression as implemented in the lm-function in R. Model control is performed by plotting the standardised residuals vs. the fitted values and normal probability plots of the standardised residuals. Standardised residuals above 3 are considered outliers and plotted on a log-transformed scale.where t, should be understood as the expected count number of a particular mRNA transcript. It is normally assumed that the expected count number is directly proportional to the number of mRNA transcripts in the sample. The coefficient of proportionality depends on e.g. the sequencing method and coverage depth. Hence, for tag-seq, it is assumed that b = 0. In the following we calculate the IDL for two models incorporating over-dispersion.For tag-seq, concentration, Y(t) is NB1 -distributed, i.e. NB1 is the linear mean-variance negative binomial distribution, with dispersion parameter θ, mean μ and variance μ + θμ. The pmf of this distribution is [First consider the linear mean-variance negative binomial distribution NB1 where ution is In this caseθ/log20(1 + θ) counts in the specific experimental setup.This implies that in order to reliably observe an mRNA transcript, the expected count for the mRNA transcript should be more than Y(t) is NB2 -distributed, i.e. NB2 is the quadratic mean-variance negative binomial distribution, with dispersion parameter θ, mean μ and variance μ + θμ2. The pmf of this distribution isNext, consider the quadratic mean-variance negative binomial distribution NB2 where In this caseθ − 1)/θ counts in the specific experimental setup.This implies that in order to reliably observe an mRNA transcript, the expected count for the mRNA transcript should be more than  for both the NB1 and NB2 model will in practice depend on the sample purity. If pThen, it is straightforward to calculate the MDL as function of tkxi, where 0 = x1 ≤ ⋯ ≤ xn ≤ 1, then, for tag-seq, this means that tk is estimated by a negative binomial regression model with log-linkThis shows that the MDL is inversely proportional to the sample purity. The IDL is estimated by dilution curves in the following way: Let the transcript be measured at the following dilutions of the k’th transcript, ak = log tk and x = 1, 2. This is done by means of maximum likelihood estimation of the parameters as implemented with the gamlss-function in R [where ion in R . Model cy = x and under random sampling it will behave like y=x2. The acceptance region at a given level of significance for the hypothesis of random sampling can be constructed by the hypergeometric distribution [We assessed the cross platform reproducibility by means of correspondence curves . A correribution .TBP as reference gene and the pure OCI-Ly8 cell line as reference sample. No reverse transcription and no template control samples were used as controls.For determination of mRNA expression levels by RT-qPCR measurements were performed in triplicates, and the average cycle thresholds (Cq) were used to determine fold-change. The mRNA expression levels were determined by the ∆∆Cq method  using TBThe authors declare they have no competing interests.MBK was responsible for the cell line culturing, flow sorting, sequencing, array analysis, RT-qPCR validation, data interpretation and drafted the manuscript. AEB implemented the tag-seq data analysis workflow, and MRD implemented the exon array data analysis workflow. AEB and MRD participated in data interpretation and paper drafting. KSB assisted with array analysis, AS with flow sorting, JSB with cell culturing and MS and SF assisted with data analysis. MN, HEJ, KLN, KD and MB conceived the study, and participated in its design and coordination and assisted in drafting the manuscript. All authors read and approved the final manuscript.Analysis workflow.Click here for fileCorrespondence curve (solid blue line) and 5% acceptance region (grey area) for the test of independence in gene selection between the exon microarray and tag-seq platforms.Click here for fileDifferentially expressed mRNA transcripts between the pure OCI-Ly8 and HEK293 samples identified by tag-seq.Click here for fileDifferentially expressed mRNA transcripts between the pure OCI-Ly8 and HEK293 samples identified by tag-seq.Click here for fileHierarchical clustering after subsampling of all cell populations based on all genes (A) exon microarray and (B) tag-seq.Click here for fileModel control for the linear, NB2 and NB1 models.Click here for fileSession information.Click here for file


Helicobacter pylori causes acute and chronic gastric inflammation induced by proinflammatory cytokines and chemokines secreted by cells of the gastric mucosa, including gastric epithelial cells. Previous studies have demonstrated that the bacterial arginase, RocF, is involved in inhibiting T cell proliferation and CD3ζ expression, suggesting that arginase could be involved in a more general dampening of the immune response, perhaps by down-regulation of certain pro-inflammatory mediators.Helicobacter pylori strain 26695, an arginase mutant (rocF-) or a rocF+ complemented strain. H. pylori infection triggered altered host gene expression in genes involved in cell movement, death/growth/proliferation, and cellular function and maintenance. While the wild type strain stimulates host inflammatory pathways, the rocF- mutant induced significantly more expression of IL-8. The results of the microarray were verified using real-time PCR, and the differential levels of protein expression were confirmed by ELISA and Bioplex analysis. MIP-1B was also significantly secreted by AGS cells after H. pylori rocF- mutant infection, as determined by Bioplex. Even though not explored in this manuscript, the impact that the results presented here may have on the development of gastritis, warrant further research to understand the underlying mechanisms of the relationship between H. pylori RocF and IL-8 induction.Global transcriptome analysis was performed on AGS gastric epithelial cells infected for 16 hours with a wild type H. pylori arginase modulates multiple host signaling and metabolic pathways of infected gastric epithelial cells. Arginase may play a critical role in anti-inflammatory host responses that could contribute to the ability of H. pylori to establish chronic infections.We conclude that  Helicobacter pylori (H. pylori) causes a spectrum of gastric diseases ranging from mild to severe gastritis and peptic ulcers to gastric cancer  hydrolyzes arginine to ornithine and urea, the latter of which may serve as an endogenous substrate for the powerful H. pylori urease enzyme, to generate carbon dioxide and ammonia. The H. pylori RocF is associated with the inner cell membrane and uses cobalt as cofactor, as opposed to mammalian arginases which use manganese [H. pylori to acid in an arginine-dependent fashion [rocF- mutant is unable to hydrolyze and consume arginine [anganese -12. Inte fashion . Moreovearginine ,14, extrarginine .H. pylori, the rocF- mutant promotes T cell proliferation and expression of the important T cell surface signaling molecule, CD3ζ [H. pylori arginase in gastric epithelial cell response is unknown. We therefore sought to determine the impact of H. pylori rocF- on epithelial cell transcription and cytokine/chemokine profiles using Illumina gene chip analysis, real-time PCR, ELISA and Bioplex analysis.Both in a tissue culture system and from peripheral blood from human volunteers, it was shown that, in contrast with wild type le, CD3ζ . Thus, ain vitro model for H. pylori interactions [H. pylori arginase, encoded by rocF, plays an important role in both innate and adaptive immunity [rocF- mutant, and rocF+ complemented H. pylori strains. The log10 transformed data of the net intensity signal, using non-infected cells (NS) as reference, was used to generate a heat-map of gene expression profiles of the different H. pylori treatments in AGS cells. As seen in Figure rocF+ was very similar. In contrast, the gene profile obtained in response to the infection with the H. pylori arginase mutant (rocF-) was completely different to the profiles generated by the other two strains as evidenced by the localization of the rocF- strain in a separate branch of the dendrogram. Interestingly, a set of genes associated with pro-apoptotic and anti-apoptotic pathways were differentially expressed in the rocF- mutant as compared to the wild type or rocF+ strains , we found that while 262 genes were common to the infection with all three H. pylori strains, infection with rocF- resulted in modulation of 2,563 genes of which 1,718 were uniquely induced by this strain  of which only 23 were uniquely induced by the WT strain and 308 by the rocF+  was assembled and showed a central core formed by the NFκB family. This central core was further expanded to highlight the most relevant genes (those with stronger associations) and this revealed a set of genes associated with inflammatory responses, including IL-8NFκB, and STATs , the latter functioning as an IL-8 activator either directly or indirectly through the activation of other genes involved in IL-8 transcription  and involved many of the genes present in the network, including IL6IL-8NFKBAP-1JUN, and IL1B (data not shown). Even though IL-8 was the most modulated gene from our microarray analysis, it was not the only one. Table H. pylori. Based on the direct analysis of the gene list, and those obtained from networks and pathways analysis, and very especially on the role of IL-8 in the induction of inflammatory responses, we focused our efforts on confirming the effects of the infection on IL-8 production.Gastric adenocarcinoma epithelial cell line AGS has been extensively studied and reviewed as a valid ractions . H. pyloimmunity ,16, but GAPDH as housekeeping and normalizing with an internal calibrator. The fold induction at 0 h was subtracted and the signal obtained in the NS used to determine the ratio of the induction of each gene in WT, rocF- and rocF+ infected AGS cells. As seen in Figure H. pylori rocF- mutant induced 40 and 23 times more IL-8 than the H. pylori WT or the rocF+ complemented strain, respectively (p < 0.0001). No significant difference was found in the fold induction of the other genes . Fourteen cytokines and growth factors were induced by at least one of the H. pylori strains. IL-8 was the most abundantly expressed cytokine/chemokine, especially by the AGS cells infected with the H. pylori rocF- mutant strain (1068 ± 243.8 pg/ml) as compared to the WT (428 ± 13.4) or the complemented isogenic strain (529 ± 73.1)  [How does arginase in wild type n (CD3ζ) . AnotherH. pylori rocF- mutant is through altered spermine produced by the AGS cells. Previous reports have shown that H. pylori infection induces ornithine decarboxylase (ODC) in macrophages [SMS), we did find that ornithine decarboxylase (ODC) and spermidine synthase mRNA (SRM), two key enzymes in the production of spermine, were 20% and 41%, respectively, reduced in the rocF-mutant-infected AGS cells (data not shown). It has also been shown that spermine can reduce the inflammatory response by post-transcriptional inhibition of the production of pro-inflammatory cytokines, including TNFα, IL6, MIP-1α, and MIP-1β [H. pylori with AGS cells, spermine levels may be elevated in the AGS cells, leading to a dampening of the chemokine/cytokine pro-inflammatory response. These possibilities await further in depth analyses.A fourth possibility is that the increase in IL-8 production induced by the rophages ,18. ODC d MIP-1β , and everocF- knockout mutant, and a rocF- complemented (rocF+) H. pylori strain, with uninfected AGS cells as a control. The first observation with the microarray analysis was an overall increase in the number of genes that participate in several signaling pathways previously investigated with H. pylori infection, notably with NFKB and AP-1 activation and mitogen-activated protein kinase  [JUN-mediated signaling. From this activation cascade, the induction of IL-8 marked the greatest difference between the rocF- mutant H. pylori versus either the WT or the rocF+ complemented strain. Our results show a significant increase of mRNA and protein levels of IL-8 in AGS cells infected with the rocF- mutant strain, suggesting that WT bacteria may be able to control the inflammatory infiltration of immune cells by controlling the production of IL-8, which is a potent chemotactic factor for inflammatory cells, especially neutrophils [We performed pair-wise comparison of transcriptome on the human adenocarcinoma gastric cell line AGS after infection with 26695 wild type, its isogenic , SAPKs) , along wtrophils -24.H. pylori factors have been suggested to stimulate IL-8 expression, including peptidoglycan, LPS, CagA, VacA, PicB, IceA, urease (and even ammonia) [H. pylori have been demonstrated, including the presence of a less potent LPS and cholesterol glycosylation [H. pylori-gastric cell interaction, and the complexity of the lesions induced by the infection, it is expected that multiple pathways are activated and the balance of those pathways may determine the presence or the evolution of the gastric lesions.While many ammonia) -28, lesssylation ; howeversylation -32. Whilsylation , the limH. pylori rocF- strains in the cellular infiltration of the gastric mucosa are currently underway. To the best of our knowledge, there is only one published work trying to measure the levels of H. pylori arginase in gastric biopsies of patients with gastritis and its correlation with disease [H. pylori arginase in biopsies but the authors were not able to establish a correlation with the degree of gastritis. The reason for the increased number of genes modulated by the rocF- H. pylori, when compared to the WT and the rocF+ bacteria, is not known; however, our results, rather than suggesting the existence of H. pylori arginase mutants in human gastric lesions, highlights the importance that this enzyme may have in the interaction of the bacteria with cells in the human gastric mucosa, and through them, with the immune system. Taken together our results suggest that H. pylori arginase, by modulating the production of IL-8 may play a significant role in the survival of H. pylori in the gastric environment. By preventing an over-zealous immune response, H. pylori can achieve its chronicity through the production of arginase and probably other bacterial factors that contribute to the overall global success of this important and highly-adapted gastric human pathogen.Efforts to determine the effect of the infection with  disease . That woH. pylori arginase in the modulation of inflammatory responses. Since IL-8 is pivotal for the infiltration of inflammatory cells into the gastric mucosa, H. pylori arginase may be involved in reducing the tissue damage associated with the evolution of the gastric lesions through the modulation of multiple pathways on the gastric epithelial cells.Our results highlight the importance of H. pylori 26695 strains  were described previously [2, 5% O2, and 10% CO2. Prior to coculture experiments, H. pylori cells were grown in Ham’s F-12 with heat-inactivated 1% (v/v) fetal bovine serum (FBS) [H. pylori growth was monitored by ATP level using Cell Titer-Glo® cell viability assay kit , as validated previously [eviously . All strum (FBS) . H. pyloeviously  and by p2. For the experiments, 1 x 106 AGS cells were seeded into 6-well plates containing 2 ml fresh F-12 supplemented with 3% heat-inactivated FBS and cultured for 8 hours. The media was replaced with 2 ml fresh F-12 containing 3% heat-inactivated FBS before inoculation of the different H. pylori at a multiplicity of infection (MOI) of 20. The infected cells were cultured for additional 16 h after which the media was collected and stored for ELISA and BioPlex analyses and the RNA extracted for microarray and real-time PCR studies.AGS gastric epithelial cells  were maintained in F-12 with heat-inactivated 10% FBS at 37°C in an atmosphere of 5% COTo extract the RNA from the AGS cells, coculture supernatants were removed by aspiration and 1 ml of TRIZOL  was added immediately to each well. RNA was extracted as recommended by the manufacturer and was stored at −80°C until further use. RNA was dissolved in DNase/RNase-free water, quantified by NanoDrop (Fisher Scientific) and set at a concentration of ~1.0 μg/μl. The quality of the RNA was confirmed by Agilent 2100 Bioanalyzer . Each experiment was repeated four times. Two hundred ng of RNA were used to make biotinylated cRNA using the Illumina TotalPrep RNA Amplification Kit , and hybridized to the Illumina chips for 14 hours at 58°C. After washing and staining, the arrays were scanned with the BeadArray Reader (Illumina Inc.) and analyzed with the GenomeStudio software (Illumina).p value for the difference at <0.05. Scatter plots comparing the non-infected cells against each one of the other treatments  were used to select only those genes with > 3 fold difference (up or down-regulated) as compared with the non-infected cells, and p values less than 0.05 (p < 0.05). In addition, the Log10 of the ratio between the normalized intensity in the infected cells and the normalized intensity in the non-infected cells was determined and used to generate heat maps.After subtracting the background, the samples were normalized assuming a similar distribution of transcript abundance in all the samples . The netFor real-time PCR (qPCR), total RNA extracts were DNase treated and reverse-transcribed with SuperScriptase III (Invitrogen) with random hexamers. TaqMan pre-designed arrays were used to check the levels of mRNA expression of IL-8, using cyclophilin A as housekeeping gene, and following the vendor’s recommendations . The 5 μl reaction was subjected to two minutes at 50°C, 10 minutes at 95°C and finally 40 cycles at 95°C for 15 seconds and 60°C for one minute in a 7900HT real-time PCR machine (Applied Biosystems). The delta Ct’s (ΔCt and ΔΔCt) and fold induction of IL-8 were determined using an internal control as calibrator.g for 5 min and then 16,000 x g for 10 min, 4°C). Manufacturer’s manuals were followed for BioPlex  and human IL-8 ELISA assay . For Bio-Plex analysis, 2 μl of anti-cytokine conjugated beads were added to each well, followed by diluted culture supernatants. After 30 min incubation, samples were washed three times with Bio-Plex wash buffer, and then 25 μl of detection antibody solution was added and incubated for another 30 min. Streptavidin-phycoerythrin  was added to each well and then washed. For hIL-8 ELISA, duplicate measurements were done for four separate experiments. Samples were read at 450 nm on an ELISA reader (Bio-Rad), of which lowest detection limit was 0.8 pg/ml .Culture supernatants were collected at indicated time points and clarified of bacteria and host cells by centrifugation . The networks generated were ranked and built according to G-scores and p values.Online computational tools of Metacore  were used to identify annotated networks of interacting genes, pathways and associated biological functions among genes profiled from the microarray analysis, using more than 700 canonical maps and pathways which are continuously being updated (t-test.All data in each experiment of ELISA and real time PCR are presented as mean ± SEM of three or four different experiments. To check for any difference between the several treatments we did a one-way ANOVA analysis. To determine differences between specific treatments we did a two-tailed unpaired SHK and RAS conducted all the experiments described in the manuscript; DJM and JZ designed the study, provided support and helped with the experiments, and co-wrote the manuscript. All authors read and approved the final manuscript.


Diamond is not only a free standing highly transparent window but also a promising carrier confinement layer for InN based devices, yet little is known of the band offsets in InN/diamond system. X-ray photoelectron spectroscopy was used to measure the energy discontinuity in the valence band offset (VBO) of InN/diamond heterostructure. The value of VBO was determined to be 0.39 ± 0.08 eV and a type-I heterojunction with a conduction band offset (CBO) of 4.42 ± 0.08 eV was obtained. The accurate determination of VBO and CBO is important for the application of III-N alloys based electronic devices. Among the group III nitrides, InN is of great interest because of its extremely high predicted electron mobility , small e19 cm-3 and 370 cm2/Vs respectively, as determined by Hall effect measurement in the InN/sapphire film.Three samples were used in our XPS experiments, namely, a 350-nm-thick InN layer grown on c-plane sapphire, a 2-mm-thick single-crystal diamond synthesized at high temperature and high pressure(HTHP), and a ~5-nm-thick InN grown on diamond. InN films in this study were grown by horizontal low-pressure metal-organic chemical vapor deposition, as reported elsewhere .The crysEF). A large number of electrons are excited and emitted from the sample during the test, so the sample is always positively charged and the consequent electric field can affect the measured kinetic energy of photoelectron. A low-energy electron flood gun was utilized to achieve charge compensation. The total energy resolution of this XPS system is about 0.5 eV, and the accuracy of the observed binding energy is within 0.03 eV after careful calibration. The measurements were as follows: first, low-resolution survey scan mode was used to determine which elements were present on the sample surfaces. Then, very-high-resolution spectra were acquired to determine the binding energy  in the survey spectra. Since only the relative energy position in each sample is needed to determine the VBO, the absolute energy calibration for a sample has no effect on the universal energy reference.The XPS measurements were performed on a PHI Quantera SXM instrument with Al Kα (energy 1486.6 eV) as the X-ray radiation source, which had been carefully calibrated utilizing work function and Fermi energy level (Ev) can be calculated from the formulaThe VBO (Δ5/2 and C1s core levels (CLs) in InN and diamond, which are measured in the InN/diamond heterojunction. 5/2 spectra for the InN and InN/diamond samples, the C1s spectra for the diamond and InN/diamond samples, and the valence band photoemission for both InN and diamond samples are shown in Figure where 5/2 core-level lineshapes are slightly asymmetric, with a high binding energy shoulder on the main peaks. This phenomenon has been reported by several groups [2 when heavily doped with Sb [2 [As illustrated in Figure l groups -22. Due l groups , the latl groups . Accordi with Sb ,24, indi with Sb  and PbO2th Sb [2 ,27 calcun is the carrier concentration, ε(∞) is the high frequency dielectric constant, and m* is the effective mass of the conduction electrons. Compared with the metals in his model, the carrier concentration in our sample surface is much lower, which means that only small energy separations exist between the screened and unscreened core-level components. This results in an asymmetric core-level XPS lineshape with just a weak high binding energy tail due to plasmon losses, which is consistent with our experimental results.Here 5/2 photoemission, the higher-binding energy component (445.27 eV) to the "unscreened" final-state peak, and the mid-binding energy component (444.21) to the In–O bonding. In Figure Based on all the arguments made above, we attribute the lower-binding energy component (443.42 eV) in Figure + bombardment with a voltage of 1 kV at a low sputtering rate of 0.5 nm/min, which alleviates damages to the sample. The reduced thickness (less than 1 nm) is calculated by the sputtering rate, and the O-related bondings were absent in cleaned InN/diamond heterojunction because of the sputtering process. In Figure In order to avoid the surface oxidation and reduce the contamination effect, the InN/diamond sample was subjected to a surface clean procedure by Ar, as is mentioned above. The VBO values can be calculated by substituting those measured values in Table Ev) is -0.39 ± 0.08 eV. The CBO (ΔEC) is given by the formula The lower-binding energy components related to "screened" final-state are chosen for VBO calculation because the peak and line width of higher-binding energy  depend on the excitation of bulk, surface plasmon, and surface treatment ,23, as i7 V/m. Assuming the heterojunction InN overlayer thickness of ~4 nm after Ar+ bombardment, the error of VBO induced by lattice mismatch is less than 60 meV. Besides, practically all nitride epitaxial layers are characterized by dense networks of threading defects extending from the substrates to the surfaces [, the strains in pseudomorphic epi-films relieve mostly, which means the "residual" effect of piezoelectric field is greatly reduced. As a result, the strain-induced piezoelectric effect can be neglected here.As XPS measurements are spatially averaged due to the finite mean free path of elastic electrons (1.5–2 nm), band bending could induce a systematic error in our measurements. Due to the lattice mismatch between InN and diamond, especially the small linear pressure coefficient of InN (~0.06 meV/GPa) , the bansurfaces , the strEv ~ 0.39 ± 0.08 eV and conduction band offset of ΔEc ~ 4.42 eV was obtained. The accurate determination of the band alignment of InN/diamond indicates that the diamond can provide an effective carrier confinement in InN/diamond based electronic devices.In summary, the valence band offset of the InN/diamond heterojunction has been measured by XPS. A type-I band alignment with a valence band offset of Δ


Pulmonary alveolar proteinosis (PAP) is a rare disorder characterised by abundant alveolar accumulation of surfactant lipoproteins. Serum levels of KL-6, high molecular weight human MUC1 mucin, are increased in the majority of patients with PAP. The prognostic significance of KL-6 in PAP is still unknown. Aim of the study was to evaluate whether serum KL-6 levels correlate with the outcome of the disease.From 2006 to 2012, we prospectively studied 33 patients with primary autoimmune PAP. We measured serum KL-6 levels by ELISA , and evaluated the correlation between initial KL-6 levels and clinical variables. Disease progression was defined as deterioration of symptoms, and/or lung function, and/or chest imaging.2, A-aDO2, DLCO, VC and TLC . The change over time of serum KL-6 correlated with the change over time of DLCO (p=0.017). The initial serum KL-6 levels were significantly higher in patients with disease progression than in those with remission (p<0.001). At a cut-off level of 1526 U/mL, the initial serum KL-6 level predicted disease progression . At a cut-off level of 2157 U/mL, the initial serum KL-6 predicted the necessity of repeated whole lung lavage . In the multivariate analysis, the initial serum level of KL-6 was the strongest predictor of disease progression .The initial serum KL-6 levels were significantly correlated with the baseline PaOSerum KL-6 seems to predict outcome in PAP. Pulmonary alveolar proteinosis (PAP), first described in 1958 -6.KL-6 is a mucin-like glycoprotein present in the human MUC1 mucin . The oriThe role of serum KL-6 as sensitive biomarker for various interstitial lung diseases has been demonstrated in idiopathic pulmonary fibrosis, radiation pneumonitis, drug-induced pneumonitis, hypersensitivity pneumonitis, CTD-associated ILD, pulmonary sarcoidosis, and cystic fibrosis -15. SeriThe clinical utility of serum KL-6 in PAP has been only partially investigated ,22-25. TWe prospectively studied 33 Caucasian PAP patients between 2008 and 2012. The study was approved by the local IRB . Informed consent was obtained from the patients.The diagnosis of PAP was based on diagnostic BAL findings, characteristic HRCT, and/or histopathologic findings on biopsy ,27. All Disease progression was defined as deterioration of symptoms , and/or lung function , and/or chest imaging (increase of the previous findings or appearance of new infiltrates characteristic of PAP) since the last follow-up visit. In order to avoid the bias of subjective interpretation in the assessment of disease progression, we included in this group with disease progression only the patients who required treatment with whole lung lavage (WLL) in the follow-up period. In our centre, PAP patients should be submitted to WLL (1) in presence of persistent (PaO2 <70 mmHg with no change for at least 3 months) or progressive respiratory failure ; (2) in the absence of respiratory difficulty at rest, the presence of exercise desaturation (decrease in PaO2 >10 mmHg or SaO2 >5%).2 > 10 mmHg since the last measurement), and chest imaging showed stability or amelioration of the previous findings.We defined a patient as improved if PAP symptoms  ameliorated or disappeared and lung function tests improved , no change in lung function tests and no new radiological infiltrates. We considered a patient as stabilized if the disease course changed from progressive to stable (see the definition above) after treatment or spontaneously.A patient was defined as being in remission, if he/she improved or remained stable and did not receive WLL within 18 months prior to the last evaluation. The 18 months limit is based on longterm experience in our centre.Serum samples were obtained by venipuncture at time of first evaluation and then every 202±148 days, and were stored at −20 or −80°C until analysis.Serum KL-6 was measured by ELISA  as described before  .Measurements including vital capacity (VC), forced expiratory volume in one second (FEV1), total lung capacity (TLC), diffusing capacity of the lung for carbon monoxide (DLCO), partial pressure of oxygen in arterial blood (PaO2), and alveolar-arterial oxygen gradient (A-aDO2) were performed along with the blood sample collection. Values were expressed as percentages of predicted normal values .t-test or Wilcoxon’s rank test for continuous variables, Chi-squared or Fischer’s exact test for categorical variables. Spearman’s or Pearson’s correlation coefficient was obtained for correlations. Receiving operating curves (ROC) analysis was used to predict disease outcome. Uni and Multivariate Cox’s proportional hazard regression model was used to analyze prognostic factors. The Kaplan-Meier method with log-rank test was used to analyze whether KL-6 levels were associated with the disease outcome. P values of <0.05 were considered statistically significant. All statistical analyses were performed using SPSS 17 .Continuous variables were evaluated for a normal distribution with the Kolmogorov-Smirnov test. Parametric data are presented as mean ± SE and nonparametric data are presented as median and interquartile range (IQR). Categorical variables are presented as either a percentage of the total or numerically, as appropriate. Comparison between 2 groups was done with Student’s 33 PAP patients were prospectively studied. The median follow-up time was 510 (90–1890) days. Demographics and disease outcome of the patients are shown in Table Mean serum KL-6 level was 2049±1893 U/mL  , inversely with PaO2 , FVC , TLC  and DLCO . The strongest correlations are shown in Figure Correlations between changes in serum KL-6 levels and changes in pulmonary function tests (PFTs) are shown in Figure ROC analysis was performed to test whether baseline serum KL-6 concentrations were predictive of disease progression, the necessity of repeated WLL, or remission. Baseline serum KL-6 concentrations were associated with disease progression and the necessity of repeated WLL Figure . ROC anaAt the cut-off level of ≥1526 U/mL, serum KL-6 levels yielded a sensitivity of 81% and a specificity of 94% to predict disease progression . A second cut-off was identified at ≥2157 U/mL with a sensitivity of 83% and a specificity of 96% to predict the necessity of repeated WLL (p<0.0001). PPV and NPV and accuracy are reported in Table To verify the utility of the cut-off for disease progression, we divided all patients into two groups; the high KL-6 group (n=14), baseline concentrations ≥1526 U/mL) and the low KL-6 group (n=19), baseline concentrations <1526 U/mL). The characteristics of the stratified patients according to the KL-6 cut-off are shown in Table Plots of the incidence of disease progression and treatment with repeated WLL for both groups were obtained using the Kaplan-Meier curve Figure . The incWe explored the predictive value of KL-6 for disease progression. In the univariate model, baseline serum KL-6 levels ≥1526 U/mL were associated with an increased risk of disease progression  Table . In the We also explored the predictive value of baseline serum KL-6 levels ≥2157 U/mL for the necessity of treatment with repeated WLL (a subgroup of 12 patients)   than serum KL-6 alone. However, serum LDH alone did not show a predictive value for repeated WLL , confirming the results of ROC analysis (see results above).This study showed that serum KL-6 levels correlate with disease progression and can predict outcome in a cohort of adult European PAP patients. At a cut-off value of 1526 U/mL for serum KL-6, the sensitivity and specificity to predict disease progression was 81% and 94%, respectively. The cut-off value of 2157 U/mL had a sensitivity of 83% and a specificity of 96% to identify patients that needed repeated WLL. Moreover, multivariate regression analysis confirmed serum KL-6 as the strongest predicting factor for disease progression and repeated WLL.It is known that serum KL-6 levels are extremely elevated in PAP patients, with mean values being much higher than those in any other ILD ,23,33,34We confirmed published data on the association of serum KL-6 with lung function tests and blood gas analyses ,10,11. PWhereas a serum KL-6 level ≥1526 U/mL predicted disease progression with a sensitivity of 81% and a specificity of 94% neither serum LDH or serum GM-CSF autoantibody did show a predictive value for disease progression. The poor correlation with severity of disease and the weak power of GM-CSF autoantibodies to predict PAP outcome are known ,30,35,36The patients with serum KL-6 levels ≥1526 U/mL had worse lung function tests than patients with lower levels, but the serum levels of LDH, GM-CSF autoantibodies and CEA did not differ significantly between the two groups. The incidence of disease progression was 80% in patients with higher serum KL-6 levels versus 15% in those with lower levels. The multivariate analysis showed that a serum KL-6 level ≥1526 U/mL, but not KL-6 as continuous variable, was associated with a 3.8 hazard of disease progression even after adjusting for covariates. The threshold of 1526 U/mL for serum KL-6 established by this study might be different in other cohorts. However, this does not impact our finding that patients with high KL-6 serum concentrations have a higher risk of disease progression than patients with lower serum levels.From previous studies it is known that 5 to 10% of PAP patients can reach a spontaneous remission ,3,30, 75According to our findings, the predictive power of serum KL-6 is high. Both disease progression and the need of repeated WLL are affected by patient age, gender, smoking history, and baseline physiology, but hazard proportional ratios for these parameters are below 2.0 and often not significant. In recent years, there was increasing interest to define accurate predictors of outcome in PAP. Up to now, the best predictors of survival have been PFTs or composite  parameters, like the disease severity score DSS ,30,38. TThere are several limitations of this study that need to be addressed. First, we did not check for the mucin-1 568 A/G polymorphism that is known to influence KL-6 serum levels ,34. In fIn conclusion, we were able to demonstrate that KL-6 is a predictive serum biomarker for the outcome of PAP. This may be of benefit in the clinical management of patients with PAP. Although our data are promising, a multicentre validation is necessary to determine whether serum KL-6 should be routinely used as a prognostic biomarker in PAP.The experiments in this study comply with the current laws in Germany.A-aDO2: Alveolar arterial oxygen gradient; BALF: Bronchoalveolar lavage fluid; CTD: Connective tissue disease; DLCO: Diffusing capacity of the lung for carbon monoxide; ELISA: Enzyme-linked immunosorbent assay; FEV1: Forced expiratory volume in one second; GM-CSF: Granulocyte macrophage colony stimulating factor; HRCT: High resolution computed tomography; ILD: Interstitial lung disease; PAP: Pulmonary alveolar proteinosis; PFTs: Pulmonary function tests; ROC: Receiver operating characteristic; TBB: Trans bronchial biopsy; TLC: Total lung capacity; VC: Vital capacity; WLL: Whole lung lavage.The authors declare that they have no competing interests.FB contributed to the conception and design of the study; collecting sample, performing biomarkers measurement, collecting, analyzing, and interpreting the data; and drafting the manuscript. SO contributed to analyzing and interpreting the data. MG contributed to measuring the biomarkers in serum and interpreting the data. MC contributed to measuring the biomarkers in serum, analyzing and interpreting the data. JG contributed to the conception and design of the study; and drafting the manuscript. UC contributed to the conception and design of the study; interpreting the data and drafting the manuscript, and he is the guarantor of the manuscript. All authors have read and approved the final manuscript.


This results in complete disappearance of the sharp Hg0 electrodissolution peak which is observed on bare Au and Au/GSH piezosensors. The multiple-peak anodic behavior of Au/CA and bare Au is replaced by a single high-field anodic peak of mercury reoxidation in the case of Au/CA-GSH sensors. The mass-to-charge plots indicate predominant ingress/egress of Hg(II) to/from the film. The strong hindrance of CA-SAM to bulk-Hg0 formation is attributed to film-stabilizing formation of surface (CA)2Hg2+ complexes with conformation evaluated by ab initio quantum mechanical calculations of electronic structure using Hartree-Fock methods. The associates CA-GSH provide an additional functionality of the side sulfhydryl group which is free for interactions, e.g. with heavy metals. It is proposed that in the film, the CA-GSH molecules can assume open (extended) conformation or bent hydrogen-bonded conformation with up to four possible internal hydrogen bonds.Multifunctional films are the basis of biosensors and play an important role in the emerging field of nanobioelectronics. In this work, films of a tripeptide glutathione (GSH) immobilized on a self-assembled monolayer of cysteamine (CA-SAM) on a quartz crystal Au piezosensor have been synthesized and characterized using electrochemical quartz crystal nanogravimetry (EQCN) with a Hg(II) ion probe. It has been found that in contrast to previously studied Au/GSH films, the Au/CA-GSH films strongly hinder the formation of Hg The c studied  and succreported . The funreported . The modreported , 15 can reported -18.Studies of GSH interactions with toxic heavy metals  are important to gain better understanding of the environmental effects on human health. In particular, the elucidation of mechanisms leading to increased susceptibility to autism , 20, diaIn this work, we have investigated the immobilization of GSH on a cysteamine-SAM (CA-SAM) formed on Au piezoelectrodes by adsorptive dissociation of a disulphide, cystamine. The change in film permeability was investigated using Hg(II) ion probe which offers rich reactivity for voltammetric analysis and large molar mass easily detectable by the electrochemical quartz crystal nanogravimetry (EQCN) , 29. The2.2.1.The electrochemical processes on a cysteamine-SAM (CA-SAM) modified Au electrode are hindered by blocking the access of electroactive species to the electrode surface. However, due to the short length of the carbon chain, the CA-SAM films are less compact and more disordered than aminothiol films with longer alkyl chains. On the other hand, the glutathione-SAM films (GSH-SAM) with longer peptide branches form gated ion channels with high ion permeability -5, 65-670 film with bulk mercury properties is formed, an extensive diffusion of Hg0 atoms into the Au substrate is observed [n is 2, 3, or nonstoichiometric.On the basis of measurements performed in this work and results of our previous studies ,4, the e V (NHE) -43,47,  are presented for the potential window from E = 0.9 to 0.2 V. It is seen that the CA-SAM decreases slightly the redox process A2 rate but does not hinder the Hg0 formation process A3. Interestingly, during the reverse anodic potential scan, the process A3′ of Hg0 oxidation to Hg22+ (cf. [2 + A4) is slightly shifted toward more positive potentials.In 2 is strongly hindered and the Hg0 reoxidation A3′ and processes A2 + A4 are virtually absent. Amazingly, the reduction Hg(II) → Hg0 remains seemingly undistorted. Almost the entire anodic reoxidation of Hg0 is concentrated in a new high potential process A5.The remarkable and unexpected change in voltammetric characteristic upon binding GSH molecules to the Au/CA film is shown in 0 formation potential (C3), one observes on a bare Au electrode  and the apparent mass change (from frequency shift measurements). In this way, with the EQCN one can always evaluate the extent of mercury reactivity and the stream of Hg ions reaching the substrate. While the maximum surface coverage by Hgad is 0.3 for AuSG films [2, for CA, which corresponds to one CA molecule per 1.5 equivalent Au surface atoms (assuming Au(111) surface with density: 2.305 nmol/cm2). The full monolayer coverage by the adsorbed Hg atoms corresponds the mass change mmono = 109.1 ng (427.4 ng/cm2). This amount of mass is not reached durig the cathodic going scan until the cathodic current peak C3 which is due to the bulk Hg0 formation. This makes the mass monitoring consistent with the known voltammetric behavior.Note that coverages were calculated using both the charge , do not indicate on any extensive engagement of the gold  at that stage.The experimental data presented in this paper, and particularly the minute mass changes observed during the initial part of the cathodic going potential scanning . Slightly lower experimental value of the slope ∂m/∂Q is due to the concomitant exchange of electrolyte ions to fulfill the Donnan equilibrium conditions at the film-solution interface. This means that the observed slope reflects the balance of mass fluxes of cation ingress and anion egress to/from the film. The major part of cation ingress is attributed to the mercury cations but small amounts of Na+ and H+ also participate in the cation uptake. The latter contribution reduces the slope because of smaller mass (per unit charge) of these cations. The egress of anions (causing a mass loss) also contributes to the lower experimental slope. The electrolyte ion fluxes associated with the electrode potential scanning can be regarded as the double-layer effect though the electrical double-layer is here rather complex due to the presence of the semipermeable multifunctional film.The analysis of the mass-to-charge ratio indicates on the ingress/egress of Hg as the major species being exchanged at the film-solution interface. This is illustrated in ca. 80 nm Au nanoparticles thus making the analysis considerably faster in comparison to micrometer sized Au particulate substrates for which the pathway for solid state diffusion of Hg atoms is substantially longer. The integration of voltammetric characteristics indicates that there is also a small imbalance of charge. It is mostly associated with the same processes that lead to the mass imbalance  intermediates to the solution is negligible due to the narrowness of the potential windows where Hg(I) ions are formed in the process C2 and A3′).Note that in all EQCN characteristics presented in 0 monolayer formation and form gradually an amorphous Au(Hg) amalgam [2OH, CN, or COOCH3.The Au-Hg alloys  are formed mainly at potentials lower than +0.52 V vs. Ag/AgCl  and they amalgam . The mas Authors , 69-77 f2.3.8-clusters per one CA molecule to reduce the calculation time. The projected film structure is depicted in 8CA structures constituting the film (part of the bottom Au atoms is not seen). The actual film structure is likely to be more disordered as no attempts have been made to account for the interplay of the solvation effects and intermolecular van der Waals forces. The CA molecules are chemisorbed on Au through the sulfur atom and with amino groups extended toward solution.The electronic structure of CA-SAM on Au has been calculated using Hartree-Fock method with 6-31G* basis set and pseudopotential for Au. The Au substrate was represented by the Au0 formation requires further elucidation. Due to the shortness of carbon backbone in cysteamine molecule which comprises only two carbon atoms, the CA-SAM is considerably more disordered than thiolate SAM's with longer alkyl chain molecules. The inherently low film order in CA-SAM's  complexes.The unusual effect of strong hindrance of CA-SAM toward the bulk-Hgacid SAM ), in add2Hg2+ is presented. The conformation of the complex is consistent with the involvement of two CA molecules adsorbed on Au in near-vertical orientation and with amino groups extended toward solution. This orientation is similar to the one observed by Raman scattering in mercaptoethanesulfonate SAM [0. The distance between two neighbor CA ligand molecules is reflected in the S-S distance which is 0.404 nm which exceeds only slightly the van der Waals distance between S atoms which is 0.363 nm. Thus, effective binding by coordination to Hg2+ cations provides framework for enhanced Au/CA film stability and increased film ordering. The reduction of Hg(II) would then comprise discharge followed by direct dissolution of Hg0 into the Au substrate (to form AunHg). In addition, the Hg0 adatoms may place-exchange with Au atom of the structure Au/CA leading to preferable configuration: Au-Hg/CA  conformation ) and L-glutathione (GSH) were purchased from Sigma Aldrich Chemical Company and used as received. Solutions were prepared using Milli-Pore Milli-Q deionized water (conductivity σ= 55 nS/cm). They were deoxygenated by bubbling with purified nitrogen.Apparatus. A standard electrochemical setup was employed for voltammetric measurements. It consisted of a Potentiostat/Galvanostat, Model PS-205B , an Electrochemical Quartz Crystal Nanobalance, Model EQCN-930, and a Data Logger and Control System, Model DAQ-716v, operating under Voltscan 5.0 data acquisition and processing software. In combined nanogravimetric and voltammetric measurements, mirror polished quartz crystal piezoresonators (QC-10Au-PB) with 5 mm diameter Au disk working electrodes, vacuum-sputtered over a 10 nm Ti underlayer, were used. The resonant frequency of these Au-piezoelectrodes was 9.975 MHz and their geometrical surface area was 0.1963 cm2, with the roughness factor R = 1.3. A standard EQCN cell with 30 mL capacity, having a side opening for sealing a quartz crystal resonator wafer (with siloxane adhesive), was used in experiments. The working electrode was polarized using a Pt wire counter electrode and its potential measured vs. a double-junction saturated (KCl) Ag/AgCl reference electrode. The interfacial mass changes were determined from the changes in oscillation frequency of the EQCN according to the Sauerbrey relationship [f) is related to the change in the interfacial mass (Δm), the piezoelectrically active area (A), the fundamental frequency (f0), which depends on the quartz properties and resonator thickness (here: 0.166 mm), the overtone number (n), and to the physical properties of quartz: its density (qd = 2.648 g cm-3) and shear modulus (qμ = 2.947×1011 g cm-1 s-2). Hence, the film mass changes Δm are directly related to the observed fundamental frequency shift:m to denote apparent mass changes derived from equations (1ab).tionship , 58:(6)periment . For theProcedures. The gold working electrodes sputter-deposited on a Ti-adhesion film on a quartz crystal wafer piezoresonator were cleaned with acetone, ethanol, and Milli-Q water, then etched in piranha solution (conc. H2SO4:H2O2 = 4:1) for 120 s, rinsed with distilled water and immediately immersed in 0.1 M NaClO4 + 1 mM HClO4 solution. The Au/CA modified electrodes were prepared by immersion of freshly prepared metal electrodes in 10 mM cystamine + 0.1 M HClO4 solution for 20 minutes which resulted in dissociative adsorption of the disulphide and formation of reproducible films. The Au/CA-GSH films were synthesized by incubating the Au electrode in 10 mM CA solution in 75% ethanol for 20 min., followed by rinsing with absolute ethanol and immersing in 10 mM GSH solution for 30 min. The electrodes were rinsed with 0.1 M NaClO4 + 1 mM HClO4 solution and immediately used for testing. The fast and effective CA-GSH interactions are attributed to the electrostatic and hydrogen bonding between the amine and carboxylate functional groups. Note that extensive hydrogen bonding has been recently discovered in thiolate-linked Au-nanoparticle networks by Zhong et al. [Econd = +0.9 V vs. Ag/AgCl) to avoid any uncontrolled mercury deposition and/or amalgam formation. The experiments were performed at room temperature, 22°C.g et al. -62. The 3, where 1 au = 0.529157 Å and 1 au3 = 6.749108 Å-3.Quantum mechanical calculations of electronic structure for adsorbed CA, CA-GSH and CA-Hg(II) complexes were performed using modified Hartree-Fock methods  with 6-34.0 phase. It is proposed that this is due to the film-ordering and stabilizing formation of surface (CA)2Hg2+ complexes with electronic structure and conformation evaluated by ab initio quantum mechanical calculations. Even stronger film-stabilizing properties provide GSH moieties in the Au/CA-GSH associates. In this situation, the anodic discharge of reduced mercury which normally presents a multi-peak structure (e.g. for bare Au or Au/GSH films), has been found to generate only one high-field peak for Au/CA-GSH piezosensors. Since the mass change associated with this peak is larger than the equivalent monolayer mass of Hg0, the anodic process must comprise electroxidation of Hg atoms from both the Au-Hg/CA-GSH associates and the underlying amalgam. The functional groups introduced to the film with tripeptide glutathione, including its free sulfhydryl group, amine, and carboxylate ligands, can be utilized in designing sensors, e.g. for heavy metals or biocompounds interacting with glutathione.The cysteamine SAM's on Au obtained by dissociative chemisorption of a disulphide, cystamine, provide sufficiently dense films that can be utilized for assembling multilayer and multifunctional sensory films. The Au/CA and Au/CA-GSH piezosensors have been synthesized and the permeability tests performed by the EQCN method indicate that the films are permeable to Hg(II) ion probe but successfully prevent the formation of a bulk-Hg


Nephrogenic systemic fibrosis (NSF) is a rare and a debilitating disease noted uncommonly in patients with impaired renal function when exposed to low-stability gadolinium-based contrast agents (Gd-CAs). According to experimental studies, cytokines released by the stimulation of effector cells such as skin macrophages and peripheral blood monocytes activate circulating fibroblasts which play a major role in the development of NSF lesions. The presence of permissive factors, presumably, provides an environment conducive to facilitate the process of fibrosis. Multiple treatment modalities have been tried with variable success rates. More research is necessary to elucidate the underlying pathophysiological mechanisms which could potentially target the initial steps of fibrosis in these patients. This paper attempts to collate the inferences from the in vivo and in vitro experiments to the clinical observations to understand the pathogenesis of NSF. Schematic representations of receptor-mediated molecular pathways of activation of macrophages and fibroblasts by gadolinium and the final pathway to fibrosis are incorporated in the discussion. Th. Thβ, vao Gd-CAs . AlthougStudies with incubated PBMC's, Hannover-Wistar rats and subtotal nephrectomized rats have confirmed the release of proinflammatory cytokines in response to exposure to Omniscan.It is thought that proinflammatory cytokines and chemokines released from tissues can attract a circulating fibrocyte which expresses CD34 and procollagen-1 . A circuCytokines when released attract circulating fibrocytes. Resident fibroblasts, bone marrow derived cells, endothelial progenitor cells, mesenchymal precursors and monocyte-derived fibrocytes may contribute to NSF.Promotion of fibrocyte differentiation from peripheral blood monocyte in the presence of gadodiamide (Omniscan) has been demonstrated in vitro. The hypothesis is that gadodiamide suppresses the inhibitory signals (serum amyloid protein) for fibrocyte differentiation .κB pathway.  Gd-CAs can stimulate collagen production from fibroblasts. Direct proliferative effects of gadodiamide (Omniscan) on in vitro NSF fibroblasts was validated by increased levels of collagen (1.8- to 4-fold) and hyaluronan (3.2- to 5-fold), change in fibroblast doubling time from 28 hrs to 22 hrs, and an increased expression of alpha-smooth muscle actin and vimentin . Similar Platelet-derived growth factor-tyrosine kinase (PDGF-TK) receptor is involved in myofibroblast proliferation and deposition of collagen. Interrupting earlier stages of PDGF-TK-receptor-mediated myofibroblast duplication is more advantageous than in the later stages when extracellular matrix has already accumulated in the fibrotic lesion. To assess the role of PDGF-TK receptor in the pathophysiology, Bhagavathula et al.  exposed Gd-CAs and free Gd have proliferative effects on collagen in vitro fibroblasts.The intracellular signaling pathways of an activated fibroblast are the MAPK and the phosphatidyl-inositol-3kinase (PI-3k). PDGF-TK receptor may be involved in fibroblast proliferation on exposure to free gadolinium.β is a profibrotic cytokine that attracts bone marrow derived fibrocytes, promotes their differentiation into myofibroblasts, increases synthesis of collagen, proteoglycans and prevents degradation of extracellular matrix by increasing TIMP-1 and plasminogen-activator inhibitor. It is secreted as native TGF-β and is proteolytically cleaved into a more active growth factor by transglutaminase. Parsons et al. [β increases gene expression of the extracellular matrix and decreases extracellular matrix proteolysis. Jiménez et al. [β mRNA expression by in-situ hybridization in the skin, fascia, striated muscle and heart biopsies of nine NSF patients. TGF-β was expressed in CD68+/Factor XIIIa dendritic cells, and may be involved in the complex process of dendritic cell maturation. They also postulated that TGF-β may be involved in the multiplication and augmentation of more antigen-presenting cells. The TGF-β pathway has also been described by Kelly et al. [β, angiotensin-converting enzyme (ACE), angiotensin II receptor-1(AT1), and p-Smad-2/3. Of the eleven biopsy specimens, 8 revealed the presence of TGF-β in the spindle cells, 8 stained for Smad-2/3, and 6 stained for both TGF-β and Smad-2/3. Faint staining of ACE was reported in 2 of the 11 specimens, while no AT1 staining was reported. All patients received at least 0.2 mmol/kg of Gd-CAs. A correlation was reported between dosage and expression of TGF-β. The authors postulated that the receptor AT1 was not involved, but other receptors in the renin-angiotensin system may play a role in NSF. A limitation of this study was that it evaluated AT1 and not AT2. It was suggested that matrix metalloproteinases, MMP-2 and MMP-9, thrombospondin-1, plasmin, and integrin alpha-v-β-6 activates TGF-β which then binds to a receptor complex leading to phosphorylation of Smad-2/3. These phosphorylated mediators then bind to Smad-4 and translocate to the nucleus. These actions can be inhibited by Smad-6 and Smad-7 (β-Smad-connective tissue growth factor (CTGF) axis in the skin biopsy specimens of 10 patients with NSF, 16 patients with systemic sclerosis, 8 patients without NSF on hemodialysis and 17 healthy controls. Patients without NSF on hemodialysis had a higher dermal expression of TGF-β mRNA and TIMP-1 in comparison to healthy controls, suggesting a profibrotic milieu in this group. The disproportionately increased levels of TGF-β, Smad2/3, CTGF, and TIMP-1 in patients with NSF suggest a profibrotic state, which may support fibroblast stimulation. Fretellier et al. [β distributed heterogeneously in the skin biopsies. The nonformulated gadodiamide group had worse skin lesions, microscopic evidence of inflammation, necrosis and extracellular matrix degradation, as well as CD34 and TGF-β1 stained cells. In contrast, the gadoterate group had no skin lesions or TGF-β staining. TGF-s et al.  performez et al.  investigy et al. . Biopsy d Smad-7 . Schierer et al.  comparedβ.Skin biopsies from patients with NSF have elevated levels of TG-2 which activates TGF-β mRNA expression has been demonstrated in the skin, fascia, striated muscle and heart biopsies of patients with NSF.Increased TGF-β, Smad-2/3, CTGF and TIMP-1 in patients with NSF suggest a profibrotic state, which may support fibroblast stimulation in skin biopsies of patients with NSF.Increased levels of TGF-β staining cells. Skin biopsies of SNx rats exposed to low stability Gd-CAs demonstrated CD34 and TGF-Metallothioneins (MTs) are cysteine rich proteins associated with cellular proliferation and programmed cell death. They are reservoirs for physiological metals such as zinc and copper and segregate potentially harmful metals such as cadmium, silver, mercury and arsenic. It is proposed that elevated levels of MT's in keratinocytes can lead to fibroblast proliferation (keloidogenesis) by inhibiting programmed cell death via zinc signaling cascades . MetalloMetallothionein gene expression is modified in the secretome of fibroblast exposed to gadodiamide.β, there was an increase in type I procollagen (4-fold) and an increase in collagen (2-fold). MMP-1 was decreased by 50% in response to TGF-β. Type I collagen levels have been shown to be elevated in the cells after treatment with Omniscan. The levels of extracellular matrix regulators may be elevated in inflammation. Kelly et al. [β stimulates TIMP-1 which inhibits matrix metalloproteinases to break down the extracellular matrix causing fibrosis. They studied 16 formalin-fixed, paraffin-embedded specimens from 10 patients with NSF for the expression of TIMP-1, MMP-1 and alpha-smooth muscle actin. It was ascertained that TIMP-1 was strongly expressed in the cytoplasm of spindle cells in all the 16 samples. 13 samples had absent MMP-1. Alpha-smooth muscle actin was not seen in the spindle cells. In a study by Perone et al. [It is well known that Gd-CAs may derange the enzyme/inhibitor system of collagen turnover. Collagen turnover is regulated by MMP-1 which breaks down extracellular matrix. MMP-1, also known as interstitial collagenase, has the capacity to cleave the triple helix of type I collagen allowing further collagen degradation. TIMP-1 antagonizes MMP-1. Alterations of collagen turnover are thought to be responsible for NSF development. An in vitro study  in whichy et al.  proposede et al. , collagee et al.  demonstrβ in skin specimens of 10 patients with NSF, 16 patients with systemic sclerosis, 8 patients without NSF on hemodialysis and 17 healthy controls. They demonstrated that decorin was overexpressed in patients with NSF compared to patients with systemic sclerosis , hemodialysis patients , and healthy controls . It also correlated with TGF-β (r = −0.72) expression. Although decorin is antifibrotic, it is ineffective in patients with NSF as there is failure of upregulation of TNF-α or SMAD-7 expression.Decorin is involved in collagen fibrillogenesis, growth factor modulation, fibrocyte differentiation and cellular growth which may perpetuate fibrosis. The role of decorin in the pathogenesis of NSF was studied by Gambichler et al. . They asα-hydroxylase and parathormone secretion. The affinity of FGF-23 to its receptor complex is strengthened by an obligate coreceptor called klotho, a transmembrane protein produced by nephrons [β receptor expression in rat's renal epithelial cells and human embryonic kidney cells [β receptor.High levels of serum phosphorus, 1, 25-dihydroxycholecalciferol (DHCC) and parathormone stimulate the release of fibroblast growth factor-23 (FGF-23) from osteocytes and osteoblasts. FGF-23, in turn, suppresses phosphate reabsorption in the kidney, inhibits 1-nephrons . The extnephrons . There inephrons . Thus, Cey cells . PatientAlthough there is evidence in support of an association between gadolinium exposure and the development of NSF in end stage renal disease (ESRD) patients, there are a considerable number of in-vitro studies that further suggest that this association also involves the role of permissive factors. It is arguably intuitive to oversimplify that the development of this disease is as simple as an exposure to a gadolinium based contrast agent. If this was indeed the case, it is reasonable to conclude that far more patients would have developed NSF than there are reported cases. Although largely unproven in human subjects yet, one may hypothesize a scenario wherein patients are likely to develop NSF after exposure to gadolinium if the right environment exists. The following discussion is an attempt to summarize the permissive factors which were initially implicated in the development of NSF lesions. Some of them have been discounted in subsequent publications . An exte+ progenitor cells [3+ ions to enter the interstitial space and tissues. Second, the associated inflammation can stimulate the production of profibrotic cytokines which can then lead to the attraction of circulating fibrocytes to tissues containing Gd3+ ions, causing an increase in the collagen production leading to fibrosis [Earlier studies linked the possibility of vascular reconstructive surgical procedures to the onset of NSF in some individuals . For exaor cells . Gd-CAs or cells . This cafibrosis . The presence of right atrial clots, thrombosed veins from indwelling catheters, thrombosed vascular accesses, pulmonary embolism , renal vP < 0.0001). They found a high rate of infection at the time of magnetic resonance imaging contrast administration in a small group of patients who later developed NSF. Furthermore, for patients without infection, the association of NSF with renal failure was also noted to be highly significant (P < 0.0001). It is believed that the presence of an infective state triggers cytokine proliferation which, in turn, stimulates circulating fibrocytes leading to NSF. However, Lauenstein et al. noted in their retrospective study that the 2 out of the 14 NSF patients with associated acute peritonitis showed no significant relationship between severe infection and NSF [Although a limited study, some investigators have hypothesized that an infection may be a predisposing factor for the development of NSF after exposure to gadolinium-containing contrast agents . In an o and NSF .A few case reports have suggested a link between NSF patients and an associated history of hepatic disease or chronic hepatitis C infection followed by liver transplantation . This as+ progenitor cells into peripheral circulation following administration of parathyroid hormone [Gadolinium can actively compete with calcium for cation binding sites on cellular membranes and can bind to and activate the extracellular calcium-sensing receptor, present on multiple cells and tissues such as fibroblasts, kidney, parathyroid glands, hepatocytes, and pancreas . In an a hormone , 83, it There has been speculation about the link between hypothyroidism and the development of NSF since hypothyroidism can increase the diffusion of gadolinium into the interstitial spaces, recruit circulating fibrocytes, and result in an increase to the exposure to free gadolinium. A significant association, however, could not be established in a multivariable analysis . Interes3+ ion from the chelate. In a study by Grobner, five NSF patients had a mean pH value of 7.29 ± 0.04, and a mean bicarbonate value of 19.5 ± 1.7 mmol/L suggesting the role of metabolic acidosis in the pathogenesis of NSF. They advocated correction of metabolic acidosis prior to administration of gadolinium-based contrast agents in patients with end-stage renal disease [3+ ion. The rates of dissociation of cyclic ligands are much slower than their linear counterparts. Although a theoretical possibility, the degree of acidosis is too small to have any significant impact on the kinetic rate of dechelation [The relationship between Gd-CAs exposure, acidosis and the development of NSF is unclear. Acidosis may, in theory, increase the susceptibility of ESRD patients to NSF since its presence is likely to promote transmetallation with the liberation of free Gd disease . The pho disease . Further disease , 87. Bashelation . Moreovehelation , 87. A d3+ and Fe3+ ions as follows:3+ may precipitate in the tissues in the form of gadolinium phosphate that could be phagocytosed by macrophages. In order to explain the increased iron levels in such patients, Swaminathan et al. [3+. Similarly, Panesar et al. [Intravenous iron, a frequently incorporated therapy in anemia protocols for ESRD patients, may interact with Gd-CAs administration and hence be considered a permissive factor for the development of NSF. There are two theories which may explain the role of iron in the development of NSF. First, there may be an exchange of ligand (L) between Gdn et al.  suggester et al.  demonstrr et al.  demonstrr et al. . Miyamotr et al. .P = 0.004) when compared with the rats treated with gadodiamide only. They also had increased levels of deposited gadolinium (P = 0.012) [P < 0.001) [EPO therapy can promote the pathogenesis of NSF in multiple ways. EPO has the ability to induce endothelial dysfunction by inhibiting dimethylarginine dimethylaminohydrolase, thereby increasing asymmetric dimethyl arginine . It also= 0.012) . When gi= 0.012) , 95, 96.= 0.012) . Swamina< 0.001) . The mec< 0.001) . With th< 0.001)  in 1997,3+ occurs via transmetallation and perhaps even more readily since the ion radius of Gd3+ is closer to that of calcium. In vivo experiments have shown rapid precipitation of free Gd following intravenous injection of gadolinium chloride where it is phagocytosed by macrophages [When competing for the gadolinium chelate, calcium ions behave similar to ferric ions in that displacement of free Gdrophages . On the rophages . 3+ from non-ionic linear Gd-CAs was noted to be 10 times higher than from their ionic linear counterparts due to the differences in their thermodynamic stability constants. Frenzel et al. [3+ in the presence of hyperphosphatemia due to renal dysfunction precipitates as gadolinium phosphate in the tissues. Experimentally, it has been shown that high doses of lanthanides (including Gd) exceed the complexing ability of plasma and form insoluble precipitates with hydroxides, phosphates and carbonates. Abraham et al. [3+ ion. To add to the controversy, it is currently unknown whether hyperphosphatemia or the phosphate binders such as sevelamer or lanthanum pose a greater risk. No specific recommendations regarding the continuation or discontinuation of these chemical agents have been made so far [3+ ions and development of NSF lesions in an ESRD patient with demonstrable insoluble gadolinium in the dermis [The role of hyperphosphatemia in the pathogenesis of NSF could be explained by two theories. First, the release of Gdl et al.  have deml et al.  have demm et al.  noticed e so far . Increase so far . Interese dermis .β. A novel theory is the process through which klotho reduction in CKD patients induces a premature aging phenotype and causes a profibrotic state. PDGF-receptor-mediated activation of fibroblast is a promising theory as PDGF receptor inhibitors may be used to target NSF lesions. Some of the aforementioned permissive factors have been implicated in in vitro studies. However, further studies in humans, which would take into account their role as a combined entity, are deemed necessary to provide a definite explanation to this rather interesting, yet complicated disease. In summary, many pathophysiological mechanisms have been proposed for NSF. Transmetallation, release of cytokines by the activation of skin macrophages, production of collagen, extracellular matrix and myofibroblasts by fibrocytes have all been demonstrated conclusively in animal experiments. There are intriguing theories of the role of decorin, metallothionein, osteopontin and gadolinium-stimulating TG-2 leading to the production of active TGF-